Synthesis and Purification of Lipid-conjugated Fluorescent pH Sensors

Lipid-conjugated pH sensors based on fluorophores coupled to lipids are a powerful tool for monitoring pH gradients in biological microcompartments and reconstituted membrane systems. This protocol describes the synthesis of pH sensors based on amine-reactive pHrodo esters and the amino phospholipid phosphatidylethanolamine. The major features of this sensor include efficient partitioning into membranes and strong fluorescence under acidic conditions. The protocol described here can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines. Graphical overview Synthesis of lipid-conjugated pH sensors based on amine-reactive fluorophore esters and the aminophospholipid phosphoethanolamine (PE)

. † Values upon coupling to DOPE. Note that the emission spectrum of SNARF-1 undergoes a pH-dependent wavelength shift, thus allowing the ratio of the fluorescence intensities from the dye at two emission wavelengths to be used for more accurate determinations of pH.

Materials and reagents
All catalog numbers provided below shall serve as guide; alternative sources can be used as well.   Glassware should be used throughout the procedure, since lipids stick to plasticware, and chloroform can extract components from plasticware. Lipid stocks are handled on ice to reduce evaporation of chloroform during pipetting. In this section, the coupling of DOPE with pHrodo Red NHS-ester will be described. The procedure was also successfully performed using pHrodo Green STP-ester and Alexa Fluor 488 NHS-ester with DPPE as lipid. The labelling of short-chain lipids such as C16C6PE is also possible using this procedure. Note: The volumes given below serve just as an orientation. All volumes need to be calculated properly for each reaction depending on the used fluorophore and lipid.      (coloured bottom phase) to a new glass tube and dry down via N 2 stream or in a desiccator. Proceed with quantification (Section D) or store at -20 °C.

C3. Purification via column chromatography
As an example, the purification of Alexa Fluor 488 DPPE is used. The exact gradient conditions can vary depending on the used lipid and fluorophore.
a. Provide the column with glass wool plug to create a tight filter in front of the valve. b. Transfer enough CM Sepharose (typically approximately 20-25 mL of suspension) into a beaker to yield a stationary phase of 10 cm height in the column (1cm diameter).
c. Fill the Sepharose into the column in one go and run out surplus MeOH.
Note: Be careful to never let the column run dry! d. Close the valve before the top of the column runs dry. To remove air bubbles in the column, tap it gently e.g., with a cork ring while the Sepharose is settling.
e. After settling, cover the Sepharose under a 1-3 mm thick layer of glass beads. Gradually rinse the MeOH to CHCl3 according to the mixtures given in Table 2. 2. Dissolve the reaction product in approximately 250 μL of CHCl3 and carefully add it to the top of the column by moving in a circle along the wall to ensure an even distribution. Wash your sample tube with small volumes until colourless and apply it on the column ( Figure 5A).
3. Perform the column chromatography while gradually increasing the MeOH content of the eluent, starting from pure CHCl3 (Table 3). Collect all fractions separately in 10 mL glass tubes.
4. Evaporate the solvent in all sample tubes either in a desiccator or with a rotary evaporator. The reaction product can be identified via TLC ( Figure 5B). Continue with quantification or store the samples at -20 °C.  D. Quantification of the reaction according to Bartlett (1959) 10. Boil all samples at 80 °C in the water bath for 10 min. 11. After cooling all the samples to room temperature, transfer each sample in duplicates to a 96-well plate. 12. Determine the absorption of each well using a microplate reader set to 812 nm. The reading can be repeated at 780 and/or 720 nm to adjust for the best sensitivity. 13. Average the duplicate and triplicate readings of each standard and sample, respectively. 14. Subtract the value of the blank standard (0 nmol phosphate) from all standard and sample readings. This is the corrected absorbance. 15. Plot standard curves (mean absorbance of wavelengths vs. nmol phosphate) and perform linear regression using e.g., Microsoft Excel, resulting in an equation of the type y = mx + b, where y is the absorbance, x is the phosphate concentration, and m is the slope; the intercept of the y-axis b is 0 after subtracting the value of the blank standard.
Published: Jun 05, 2023 16. Use the regression line to solve for sample concentration, by comparing the sample absorbance to the standard curve obtained ( Figure 6). 17. Analyse your product using reliable methods for small amounts such as mass spectroscopy. A typical result is shown for pHrodo Green-labelled DOPE in Figure 7. Since no chemical structure was published by the manufacturer and only an approximate molecular weight of ~750 g/mol was given, indicated signals correspond to the expected product peak in the range of mass per charge (m/z) 1 ,250.